Polynucleotides encoding the anti-PEM antibody BW835 variable domains and expression vectors

ABSTRACT

The invention relates to polynucleotides encoding the BW 835 variable domain of an antibody against a tumor-associated antigen which is mainly derived from tumors from the group of carcinomas of the breast, ovaries and prostate, as well as adenocarcinomas of the lung, which additionally react with polymorphic epithelial mucin (PEM), and expression vectors containing the polynucleotides.

This is a division of application Ser. No. 07/957,827, filed Oct. 8,1992, now abandoned.

The invention relates to monoclonal antibodies against atumor-associated antigen which is mainly derived from tumors from thegroup of carcinomas of the breast, ovaries and prostate, as well asadenocarcinomas of the lung, which additionally react with polymorphicepithelial mucin (PEM), to the preparation and use thereof and to theuse of the epitope defined by the antibody for diagnosis and therapy.

Hybridoma technology has made it possible to prepare specific monoclonalantibodies (MAbs) even against unpurified antigens. This fact has madeit possible to identify a large number of tumor-associated antigens(TAAs) which occur on certain human tumors but also on normal humantissues. Examples of such TAAs are CEA (carcinoembryonic antigen), N-CAM(neural cell adhesion molecule) and PEM (polymorphic epithelial mucin).

CEA is mainly expressed on adenocarcinomas of the gastro-intestinaltract, N-CAM is located on tumors derived from neuroectoderm, and PEMoccurs mainly on carcinomas of the breast and ovaries. The TAAs whichhave just been mentioned are high molecular weight glycoproteins whichcarry a large number of immunogenic epitopes for the murine immunesystem. Comparative immunohistochemical investigations on cryopreservedhuman tissues demonstrate that the specificity of an MAb whichrecognizes with its idiotype (V region) an epitope I on a TAA may show adifferent tissue binding than an MAb which recognizes an epitope II(Buchegger et al. (1984), Int. J. Cancer 33:643-649).

Multifarious reasons are possible for this observation: crypticity ofepitopes in certain tissues, cross-reactive epitopes on differentantigens, changes in conformation of antigens on secretion from tissuesinto the plasma (Bosslet et al. (1988), Eur. J. Nucl. Med. 14:523-528)etc. It may be concluded from this that the specificity of an MAb is notunambiguously given by the definition of the recognized antigen but isgiven by the-exact description of the V region of the MAb in conjunctionwith its immunohistochemical specificity for cryopreserved human tissuesand its serum specificity with circulating TAA structures in human serumor plasma. Thus, for example, a number of MAbs against the PEM which wasisolated by Shimizu from human milk, (Shimizu, M. and Yamauchi, K.(1982), J. Biochem. 91, 515-524) have been developed and bind todifferent epitopes and, accordingly, have different properties (Girlinget al. (1989), 43, 1072-1076, Taylor-Papadimitriou et al. (1981), Int.J. Cancer, 28, 17-21).

Certain anti-PEM MAbs (HMFG 1,2) show a strong reaction with humancarcinomas of the breast and ovaries but also react significantly withnormal human tissues (Taylor-Papadimitriou et al. (1981)). Other MAbs(SM 3, Girling et al. (1989)) react more weakly and heterogeneously withcarcinomas of the breast and ovaries but, on the other hand, do not bindsignificantly to normal human tissue.

We have succeeded, surprisingly, in producing an anti-PEM MAbwhichreacts strong with carcinomas of the breast, ovaries and prostate, aswell as adenocarcinomas of the lung, binds only weakly to normal humantissue and, in addition, is able to detect PEM very specifically inhuman serum or plasma. Methods for the immunochemical determination ofantigen are known to the person skilled in the art (see Gosling (1990),Clin. Chem. 36/8, 1408-1427). In this connection a distinction is madeessentially between two classes: homogeneous assays such as, forexample, particle-enhanced nephelometry or turbidimetry andheterogeneous methods, also called solid-phase assays.

Solid-phase assays are designed so that the analyte antigen isimmobilized out of the sample to be investigated by a trapping antibodybound to a solid phase, and the immobilized antigen is detected by asecond antibody provided with a detectable labeling moiety (conjugate).Detectable labels of this type are known to the person skilled in theart, and examples are enzymes, chemiluminescent orelectrochemiluminescent, radioactive or else colored labels.

The hybridoma cell line BW 835 which produces the monoclonal clonalantibody BW 835 was deposited on Oct. 11, 1991, at DSM, DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b,D-3300 Braunschweig, under the number DSM ACC2022.

Antibodies within the meaning of this invention also mean antibodyfragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is the nucleic acid sequence of BW 835 V_(H) (Seq. ID No. 2) andthe amino acid sequence of BW 835 V_(H) (Seq. ID No. 1).

FIG. 1b is the nucleic acid sequence of BW 835 V_(K) (Seq. ID No. 4) andthe amino acid sequence of BW 835 V_(K) "(Seq. ID NO. 3).

FIG. 2 shows the cloning of plasmid A which harbors a human IgG₃ ΔC genein the H1, H2, and H3 exons have been deleted. See Example 1.

FIG. 3 shows the cloning of plasmid B. See Example 2.

FIG. 4 shows the cloning of plasmid C. See Example 3.

FIG. 5 shows the cloning of plasmid D. See Example 4.

FIG. 6 shows the cloning of plasmid D to obtain plasmid E. See Example5.

FIG. 7 shows the isolation of plasmid F. See Example 6.

FIG. 8 shows the cleavage of plasmid F to obtain plasmid G. See Example7.

FIG. 9 shows the cleavage of plasmid E to obtain plasmid H. See Example8.

FIG. 10 shows the partial cleavage of clone H to obtain plasmid I. SeeExample 9.

FIG. 11a shows the restriction cleavage sites for MAb BW 835 V_(H). SeeExample 12.

FIG. 11b shows the restriction cleavage sites for MAb BW 835 V_(K). SeeExample 12.

FIG. 12 shows the isolation of plasmids K1 and K2. See Example 10.

FIG. 13 shows the expression vector L1. See Example 11.

FIG. 14 shows the expression vector L2. See Example 11.

FIG. 15 compares the concentration of antigen bound by Mab BW 835 inserum or plasma of patients with malignant tumors with that in healthypatients. See Example 12.

FIG. 16 compares the use of a peroxidase-labelled DF₃ antibody conjugatewhich recognizes further epitopes to the tumor-associated antigensdefined by MAb BW 835 in tumor sera, normal sera, and sera from patientswith benign diseases. See Example 13.

The preparation and the properties of this MAb (BW 835) are describedbelow:

The MAb was generated by immunizing Balb/c mice with the MCF-7 andSW-613 breast carcinoma cell lines by methods known from the literature(EP-A2-0 141079).

The distribution of the epitope defined by the MAb BW 835 oncryopreserved human carcinomas and normal human tissues is shown inTables Ia and b respectively, comparing with the MAb SM-3 (Girling etal. 1989). The data are based on an immunohistochemical detection withthe APAAP technique (Cordell at al. (1984), J. Histochem. Cytochem. 32,219). It can clearly be seen that MAb BW 835 reacts strongly with all 15carcinomas of the breast from 15 tested carcinomas of the breast,whereas the MAb SM-3 detects only 11 of 15 carcinomas of the breast.

In the case of carcinoma of the ovaries, the MAb BW 835 reacts stronglywith 6 of 8 tested tumors, and the MAb SM-3 reacts with a few cells in 6of 8 carcinomas. With all the other carcinoma types tested, especiallythe adenocarcinomas of the lung and the carcinomas of the prostate, MAbBW 835 shows a quantitatively stronger reaction. These data show thatMAb BW 835 detects more carcinomas with a quantitatively strongerreaction than the MAb SM-3 disclosed in the literature.

The binding of MAb BW 835 to normal human tissue is shown in Table Ib.The APAAP technique was employed to obtain the data in this case too.The epitope defined by MAb BW 835 is expressed significantly on theductal epithelium of the breast and the ductal epithelium of thepancreas, and is weakly expressed on the surface epithelium of thelungs, on some nerve fibers and on the collecting tubules and theglomeruli of the kidney. All the other tested normal tissues arenegative.

Because of its high selectivity, the MAb BW 835 can also be used asinducer for internal image anti-paratope MAbs. MAbs of this type mightbe employed as epitope vaccine for the therapy of human tumors.

The demonstration that the epitope defined by the MAb BW 835 is locatedon the PEM defined by MAb SM-3 was checked by a double-determinantassay.

                                      TABLE Ia                                    __________________________________________________________________________    Binding of the MAb BW 835 to cryopreserved human tumors                       __________________________________________________________________________    Human tumor type                                                                  Carcinomas                                                                          Ovarian                                                                             Prostate                                                                            Stomach                                                                             Colon Pancreas                                        of the breast                                                                       carcinomas                                                                          carcinomas                                                                          carcinomas                                                                          carcinomas                                                                          carcinomas                                  MAb pos./total                                                                          pos./total                                                                          pos./total                                                                          pos./total                                                                          pos./total                                                                          pos./total                                  __________________________________________________________________________    BW 835                                                                            15/15  6/6  4°/5                                                                         4*/6  3*/4  4*/7                                        SM 3                                                                              11/15 6*/8  1°/5                                                                         3*/6  1°/4                                                                         3°/7                                 __________________________________________________________________________    Lung carcinomas                                                               MAb  SCLC pos./total                                                                       adeno pos./total                                                                     Squamous cell pos./total                                                                 Large cell pos./total                          __________________________________________________________________________    BW 835                                                                             4*/10   11°/12                                                                        5*/11      7°/11                                   SM 3 2*/10    6*/12 4*/11      3*/11                                          __________________________________________________________________________     *a few cells positive                                                         °a few areas of secreted products positive                        

Table I b

Binding of the MAb BW 835 to cryopreserved normal human tissue

Tissue type:

Mammary gland

Positive reaction with the acinar epithelium, positive apical stainingof the epithelium in the ducts and in some secreting vesicles

Ovary

Negative

Pancreas

Positive apical staining in the ducts

Liver

Negative

Spleen

Negative

Colon

Negative

Stomach

Mucosa and some mucin-containing ducts with positive reaction

Lung

Surface epithelium of the lung with weak positive reaction

Kidney

Some glomeruli weakly stained, positive apical staining of thecollecting tubules

Table I b continued

Brain

Negative

Peripheral nerve

Some nerve fibers weakly stained

Bone marrow

Negative

Peripheral blood components

Lymphocytes, monocytes, granulocytes, erythrocytes, platelets arenegative

The MAb BW 835 used as trap was able to trap from cell culturesupernatants of the T47 cell line an antigen which was detectable by theenzyme-labeled MAb SM-3. Furthermore, in the Western blot both MAbsstain molecules which correspond to the molecular weight position ofPEM.

Once the immunological specificity data for the MAb BW 835 were defined,the mRNA was isolated from 10⁸ hybridoma cells which secrete the MAb BW835, the V genes of the heavy and light chains of the MAb BW 835 wereisolated by the method described by Orlandi et al. (1989), Proc. Natl.Acad. Sci. USA: 86, 3833-3837, and the nucleic acid sequence of theessential regions of the V gene exons were determined by the methoddescribed by Sanger et al. (1977), Proc. Natl. Acad. Sci., USA: 74,5463-5467 (FIGS. 1a, b) (Seq ID Nos: 1-4).

On repeated high-dose administration of MAbs of murine origin, such as,for example, the MAb BW 835, for in vivo therapy of humans it ispossible to immunize the patients. They are able to produce humananti-mouse immunoglobulin antibodies (HAMA) after about 10-14 days(Miller et al., (1983), Blood, 62, 988; Joseph et al., (1988), EuropeanJournal of Nuclear Medicine, 14, 367). These HAMAs may have unfavorableeffects on the pharmacokinetics and pharmacodynamics of the MAb andimpede continuation of the treatment.

In order to reduce the immunogenicity of xenogenic antibodies as far aspossible, a technique in which only the CDR loops of the V_(L) and V_(H)domains of the xenogenic antibodies are transferred to V_(L) and V_(H)domains of human antibodies has been developed (Jones, P. T., et-al.,(1986), Nature, 321, 522) (EP-A-87302620, G. Winter), and this processis called "humanization" and takes piece at the level of the V_(H) andV_(L) genes.

The technical procedure for humanization of an antibody is dividedessentially into three sections: the cloning and nucleic acid sequenceanalysis of the specific V_(H) and V_(L) genes, the computer-assisteddesign of the synthetic oligonucleotides for the transfer of the CDRloops to the human V_(H) and V_(L) domains and the transfer of the CDRloops to human V_(H) and V_(L) domains by specific mutagenesis(Rieckmann, L., et al., (1988), Nature, 332, 323; Verhoeyen, M., et al.,(1988), Science, 239, 1534).

Humanization of this type can also be carried out on MAb BW 835 in orderto improve its usability in vivo. This would entail the authentic CDRregions of the BW 835 V_(H) and V_(L) domains (defined by Kabat, E. A.,et al. (1987) Sequences of Proteins of Immunological Interest, fourthedition, US Dept. of Health and Human Services, US Government PrintingOffice) or CDR regions with a few modified amino acids being transferredto human V_(H) and V_(L) domains, it being possible for a few aminoacids of the framework regions located between the CDR regions to betaken over from the mouse antibody to the humanized antibody in order tominimise the change in the antigen-binding properties of the resultingMAb BW 835 in the humanized form.

The variable domains of the humMAb BW 835 are accordingly composed ofthe framework regions, which are authentic or modified at a few points,of the variable domains of a human MAb onto which the CDR regions whichare authentic or have been modified at a few amino-acid positions of themouse MAb BW 835 have been transplanted.

The following examples describe the steps necessary for cloning andnucleic acid sequence analysis of the V genes and for the expression ofBW 835 specificity as chimeric MAb. The techniques used in Examples 1-12were, unless otherwise indicated, taken from Molecular Cloning, aLaboratory Manual; Sambrook, Fritsch, Maniatis; Cold Spring HarborLaboratory, 1982 (pages 11 -44, 51-127, 133-134, 141, 146, 150-167, 170,188-193, 197-199, 248-255, 270-294, 310-328, 364-401, 437-506) and fromMolecular Cloning, A Laboratory Manual, Second Edition; Sambrook,Fritsch, Maniatis; Cold Spring Harbor Laboratory Press, 1989 (pages16.2-16.22, 6.30-16.40, 16.54-16.55).

EXAMPLE 1

The plasmid clone 54.1.24 which harbors the human IgG₃ Δ C gene (FIG. 2)(DE-A1-38 25 615, FIG. 2) was cleaved with PstI. The vector resultingfrom this was ligated to the largest of the resulting PstI insertfragments and transformed into bacteria. The plasmid clone A whichharbors a human IgG₃ Δ C gene in which the H1, H2 and H3 exons have beendeleted (IgG₃ Δ) was identified by restriction analysis and nucleic acidsequence determination.

EXAMPLE 2

The plasmid clone A was cleaved with HindIII and EcoRI, the ends werefilled in with Klenow polymerase, the IgG₃ Δ insert was isolated andligated into a pUC19 vector cleaved with SstI and provided with bluntends with the aid of T₄ polymerase. A plasmid clone B in which the IgG₃Δ gene is orientated so that the HindIII cleavage site is located at the5'end and the EcoRI cleavage site is located at the 3'end of the pUC19polylinker was identified by restriction mapping and nucleic acidsequence analysis (FIG. 3).

EXAMPLE 3

The plasmid clone B was cleaved with EcoRI and HindIII, the IgG₃ Δinsert was isolated and ligated into a KS+ plasmid vector (pBluescriptIIKS+; Stratagene, La Jolla, Calif.) likewise cleaved with HindIII andEcoRI. The plasmid clone C in which the IgG₃ Δ gene is flanked at the 5'and at the 3' end by a BamHI cleavage site was isolated (FIG. 4).

EXAMPLE 4

The plasmid clone c was cleaved with BmmHI, the IgG₃ Δ insert wasisolated and ligated into the expression vector pABStop (Wirth et al.(1988), Gene, 73,419-426) likewise cleaved with BamHI. The expressionplasmid D which contains the IgG₃ Δ C gene in the orientation shown inFIG. 5 was identified. In this cloning the pABStop vector loses thepolyadenylation signal and SV40 stop located between the two BamHIcleavage sites.

EXAMPLE 5

The expression plasmid D was partially cleaved with BamHI, the ends werefilled in with Klenow polymerase and religated. The expression plasmid Ein which the BamHI cleavage site 3' from the IgG₃ Δ gene is destroyedwas isolated (FIG. 6).

EXAMPLE 6

The human C kappa gene (Hieter et al. (1982), J. Biol. Chem., 257, No.3, 1516-1522) was obtained as EcoRI fragment cloned in pBR 322 fromProf. P. Leder, Harvard Medical School. The pBR322 vector was cleavedwith EcoRI, the EcoRI cleavage sites were filled in, the C kappa insertwas isolated and ligated into a pUC19 vector cleaved with SmaI. Theplasmid clone F in which the C kappa gene is flanked at the 5' end by aHindIII after a BamHI cleavage site and at the 3' end by an EcoRIcleavage site was isolated (FIG. 7).

EXAMPLE 7

The plasmid clone F was cleaved with HindIII and EcoRI, the C kappainsert was isolated and cloned into a HindIII/EcoRI-cleaved KS+ plasmid.The plasmid clone G in which the C kappa insert is flanked at the 5' andat the 3' end by a BamHI cleavage site was isolated (FIG. 8).

EXAMPLE 8

The plasmid clone G was cleaved with BamHI, the C kappa insert wasisolated and cloned into a pAB stop vector cleaved with BamHI. The cloneH in which the C kappa gene is orientated so that the HindIII cleavagesite of the pAB stop vector is located at its 5' end was identified byrestriction mapping and nucleic acid sequence analysis (FIG. 9).

EXAMPLE 9

The clone H was partially cleaved with BamHI, the restriction ends werefilled in and religated. The clone I in which the BamHI cleavage site 3'of the C kappa gene is destroyed was identified by restriction mapping(FIG. 10).

EXAMPLE 10

The V_(H) and V_(K) genes of the MAb BW 835 were amplified using the PCRtechnique and specific oligonucleotides by the method of Orlandi et al.(1989) and cloned in KS+ vectors (Gussow and Seemann (1991), Methods inEnzymology, Vol. 203) which contained V_(H) and V_(K) genes withsuitable restriction cleavage sites (FIG. 11 a for V_(H) amd b forV_(K)). The clones K1 and K2 which contain the V_(H) (K1) and V_(K) (K2)genes of the MAb BW 835 were isolated (FIG. 12).

EXAMPLE 11

The nucleic acid sequences of the V_(H) and V_(K) genes of the MAb BW835 from the clones K1 and K2 were determined by the method of Sanger etal. (1977) (FIGS. 1a, b) (SEQ ID Nos 1-4. It is possible to generatemimetics based on the CDRs from this sequence by the method described bySaragovi et al. (Saragovi et al. (1991), Science 253, 792-795).

Furthermore, the V_(H) and V_(K) gene inserts were cut out of the clonesK1 and K2 with the aid of the restriction endonucleases HindIII andBamHI and were cloned into the vectors D (V_(H)) and I (V_(K)) likewisecleaved with HindIII and BamHI. The expression vectors L1 and L2 whichcontain immunoglobulin heavy (L1) (FIG. 13) and light (L2) (FIG. 14)chain genes with the V genes of MAb BW 835 were isolated. The expressionvectors L1 and L2 can be used for the expression of a chimeric MAb withthe specificity of MAb BW 835. Examples 12 and 13 are intended toexplain the use of the MAb BW 835 for serodiagnosis of malignant tumors.

EXAMPLE 12

The MAb BW 835 was bound by adsorption to the walls pf wells ofmicrotiter plates (NUNC) by methods known to the person skilled in theart (Tijssen, P., "Practice and theory of enzyme immunoassay" Elsevier(1988), 297-328). 10 μl of sample were pipetted into each of the wellsprepared in this way and each containing 100μl of buffer (OSND,Behringwerke) and incubated at +37° C. for 2 hours. After washing threetimes with diluted Enzygnost washing buffer (OSEW, Behringwerke), 100 μlof MAb BW 835 (1 μg/ml) which was conjugated to peroxidase by knownmethods were introduced into each of the individual wells. The following2-hour incubation step at +37° C. was completed by a cycle of threewashes. Subsequently, for the third incubation step at room temperature,100 μl of a buffer/substrate chromogen solution (OUVG/OUVF,Behringwerke) were pipetted into each of the wells, and the enzymereaction was stopped after 30 minutes with 100 μl of Enzygnost stopsolution (OSFA, Behringwerke). The extinction of the samples wasdetermined as 450 nm.

Result:

The extinctions determined in this way correspond in the level thereofto the concentration of the antigen in the sample. The concentration ofthe antigen defined by the specific binding of MAb BW 835 in serum orplasma of patients with malignant tumors is distinctly raised bycomparison with that in healthy patients (FIG. 15). This particularlyapplies to patients with late-stage carcinoma of the breast, but also,surprisingly, to those with early-stage carcinoma who, with othercommercial tumor marker tests for detecting breast cancer-associatedantigens in serum (for example CA 15-3), by comparison givefalse-negative findings significantly more often, and thus overallbetter sensitivities were found for the homologous version described.

EXAMPLE 13

It is also possible to use for the detection of PEM in serum in thedouble-determinant assay in combination with the MAb BW 835 otherperoxidase-labeled antibodies which recognize further epitopes on thetumor-associated antigens defined by MAb BW 835. To do this, forexample, a test analogous to Example 12 was carried out using the DF3antibody disclosed in the literature (Kufe et-al. (1984), Hybridoma 3,223) in peroxidase-labeled form (CA 15-3-Test, Boehringer Mannhelm) asconjugate component.

RESULT

The use of, for example, DF3-POD as conjugate component to supplementthe solid-phase-bound BW 835 produced distinctly higher serum values forthe tumor sera compared with a normal serum pool and patients withbenign diseases, which once again underlines the potential of the MAb BW835 as specific component for a tumor marker test (FIG. 16).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       LeuGlnSerLeuArgAlaLeuValGlnProGlyGlySerMetLysLeu                              151015                                                                        SerCysValAlaSerGlyPheThrPheSerAsnTyrTrpMetAsnTrp                              202530                                                                        ValArgGlnSerProGluLysGlyLeuGluTrpValAlaGluIleArg                              354045                                                                        LeuLysSerAsnAsnTyrAlaThrHisTyrAlaGluSerValLysGly                              505560                                                                        ArgPheThrIleSerArgAspAspSerLysSerSerValTyrLeuGln                              65707580                                                                      MetAsnAsnLeuArgAlaGluAspThrGlyIleTyrTyrCysIleArg                              859095                                                                        GluThrValPheTyrTyrTyrAlaMetAspTyrTrpGlyGlnGlyThr                              100105110                                                                     ThrValThr                                                                     115                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 345 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTGCAGAGTCTGAGAGCCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCC60                TCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGG120               CTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAG180               TCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAA240               ATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTATCAGGGAGACGGTTTTT300               TATTACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACC345                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlnLeuThrGlnSerProProSerValProValThrProGlyGluSer                              151015                                                                        ValSerIleSerCysArgSerSerGlnSerLeuLeuHisGlyAspGly                              202530                                                                        AsnThrTyrLeuTyrTrpPheLeuGlnArgProGlyGlnSerProArg                              354045                                                                        LeuLeuIleTyrArgMetSerAsnLeuAlaSerGlyValProAspArg                              505560                                                                        PheSerGlySerGlySerGlyThrAlaPheThrLeuArgIleSerArg                              65707580                                                                      ValGluAlaGluAspValGlyValTyrTyrCysMetGlnHisLeuGlu                              859095                                                                        TyrProPheThrPheGlyGlyGlyLysValGluIle                                          100105                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 325 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CAGCTGACCCAGTCTCCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCC60                TGCAGGTCTAGTCAGAGTCTCCTGCATGGTGATGGCAACACTTACTTGTATTGGTTCCTG120               CAGAGGCCAGGCCAGTCTCCTCGGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGA180               GTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGA240               GTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACG300               TTCGGAGGGGGCAAGGTGGAGATCA325                                                  __________________________________________________________________________

We claim:
 1. A polynucleotide consisting of SEQ ID NO: 2 or SEQ ID NO:4, wherein the polynucleotide encodes the heavy chain variable region orlight chain variable region of the monoclonal antibody BW 835 which isproduced by the hybridoma cell line having accession number DSM ACC2022.2. The polynucleotide according to claim 1 consisting of SEQ ID NO: 2wherein the polynucleotide encodes the heavy chain variable region ofthe monoclonal antibody BW
 835. 3. The polynucleotide according to claim1 consisting of SEQ ID NO: 4 wherein the polynucleotide encodes thelight chain variable region of the monoclonal antibody BW
 835. 4. Apolynucleotide encoding a polypeptide comprising the amino acid sequenceconsisting of SEQ ID NO: 1 of the heavy chain variable domain ofmonoclonal antibody BW
 835. 5. A polynucleotide encoding a polypeptidecomprising the amino acid sequence consisting of SEQ ID NO: 3 of thelight chain variable domain of monoclonal antibody BW
 835. 6.Polynucleotides encoding the heavy and light chain polypeptides of amodified antibody, wherein one polynucleotide encodes a polypeptidecomprises the CDK regions of the heavy chain variable region of BW 835represented by SEQ ID NO: 1 and wherein a second polynucleotide encodesa polypeptide comprises the CDR regions of the light chain variableregion of BW 835 represented by SEQ ID NO: 3, wherein the CDR regionsare grafted onto a human framework and wherein the modified antibody isimmunoreactive with a polymorphic epithelial mucin (PEM) antigenassociated with tumors selected from the group consisting of breastcarcinomas, ovarian carcinomas, prostrate carcinomas and lungadenocarcinomas.
 7. The polynucleotides according to claim 6 wherein theframework region is modified in order to minimize the change in antigenbinding properties of antibody BW
 835. 8. An expression vectorcomprising a polynucleotide according to any one of claims 1, 2, 3, 4,5, 6 or 7.